The targeting of cytotoxic or other moieties to specific cell types has been proposed as a method of treating diseases such as cancer. Various toxins including Diphtheria toxin and Pseudomonas exotoxin A have been suggested as potential candidate toxins for this type of treatment. A difficulty of such methods has been the inability to selectively target specific cell types for the delivery of toxins or other active moieties.
Anthrax toxin is composed of three separate proteins produced by Bacillus anthracis: lethal factor (LF) (SEQ ID NOS: 1 and 2), edema factor (EF), and protective antigen (PA) (SEQ ID NOS: 3 and 4) (Leppla, S. H. Alouf, J. E. and Freer, J. H., eds. Sourcebook of Bacterial Toxins Academic Press, London 277-302, 1991). The three proteins are individually nontoxic, and become toxic only when administered in pairwise combinations. PA (83 kDa) binds to a specific cell receptor and is then cleaved by a cell surface protease which releases an amino-terminal 19-kDa fragment (Singh et al. J. Biol. Chem. 264:19103-19107, 1989). Removal of this fragment from PA exposes a high-affinity binding site for LF and EF on the receptor-bound 63-kDa carboxyl-terminal fragment (PA63). The complex of PA63 and LF or EF enters cells and probably passes through acidified endosomes to reach the cytosol.
The genes for each of the three anthrax toxin components have been cloned and sequenced. This showed that LF and EF have extensive homology in amino acid residues 1-300. Since LF and EF compete for binding to PA63, it is highly likely that these amino-terminal regions are responsible for binding to PA63. Direct evidence for this was provided in a recent mutagenesis study (Quinn et al. J. Biol. Chem. 266:20124-20130, 1991); all mutations made within amino acid residues 1-210 of LF led to decreased binding to PA63. The same study also suggested that the putative catalytic domain of LF included residues 491-776 (Quinn et al., 1991). In contrast, the location of functional domains within the PA63 polypeptide is not obvious from inspection of the deduced amino acid sequence. However, studies with monoclonal antibodies and protease fragments (Leppla, 1991) and subsequent mutagenesis studies (Singh et al. J. Biol. Chem. 266:15493-15497, 1991) showed that residues at and near the carboxyl terminus of PA are involved in binding to receptor.
Prior work had shown that the carboxyl terminal PA fragment (PA63) can form ion conductive channels in artificial lipid membranes (Blaustein et al. Proc. Natl. Acad. Sci. U.S.A. 86:2209-2213, 1989; Koehler, T. M. and Collier, R. J. Mol. Microbiol. 5:1501-1506, 1991), and that LF bound to PA63 on cell surface receptors can be artificially translocated across the plasma membrane to the cytosol by acidification of the culture medium (Friedlander, A. M. J. Biol. Chem. 261:7123-7126, 1986). Furthermore, drugs that block endosome acidification protect cells from LF (Gordon et al. J. Biol. Chem. 264:14792-14796, 1989; Friedlander, 1986; Gordon et al. Infect. Immun. 56:1066-1069, 1988). The mechanisms by which EF is internalized have been studied in cultured cells by measuring the increases in cAMP concentrations induced by PA and EF (Leppla, S. H. Proc. Natl. Acad. Sci. U.S.A. 79:3162-3166, 1982; Gordon et al., 1989). However, because assays of cAMP are relatively expensive and not highly precise, this is not a convenient method of analysis. Internalization of LF has been analyzed only in mouse and rat macrophages, because these are the only cell types lysed by the lethal toxin.
Pseudomonas exotoxin A (PE) is a toxin for which a detailed analysis of functional domains exists. The sequence is deposited with GenBank. Structural determination by X-ray diffraction, expression of deleted proteins, and extensive mutagenesis studies have defined three functional domains in PE: a receptor-binding domain (residues 1-252 and 365-399) designated Ia and Ib, a central translocation domain (amino acids 253-364, domain II), and a carboxyl-terminal enzymatic domain (amino acids 400-613, domain III). Domain III catalyzes the ADP-ribosylation of elongation factor 2 (EF-2), which results in inhibition of protein synthesis and cell death. Recently it was also found that an extreme carboxyl terminal sequence is essential for toxicity (Chaudhary et al. Proc. Natl. Acad. Sci. U.S.A. 87:308-312, 1990; Seetharam et al. J. Biol. Chem. 266:17376-17381, 1991). Since this sequence is similar to the sequence that specifies retention of proteins in the endoplasmic reticulum (ER) (Munro, S. and Pelham, H. R. B. Cell 48:899-907, 1987), it was suggested that PE must pass through the ER to gain access to the cytosol. Detailed knowledge of the structure of PE has facilitated use of domains II, Ib, and III (together designated PE40) in hybrid toxins and immunotoxins.
A single-chain antibody (sFv) consists of an antibody light chain variable domain (V.sub.L) and heavy chain variable domain (V.sub.H), connected by a short peptide linker which allows the structure to assume a conformation capable of binding to antigen. In a diagnostic or therapeutic setting, the use of an sFv may offer attractive advantages over the use of a monoclonal antibody (MoAb). Such advantages include more rapid tumor penetration with concomitantly low retention in non-targeted organs (Yokota et al. Cancer Res 52:3402.1992), extremely rapid plasma and whole body clearance (resulting in high tumor to normal tissue partitioning) in the course of imaging studies (Colcher et al. Natl. Cancer Inst. 82:1191,1990; Milenic et al. Cancer Res. 51:6363, 1991), and relatively low cost of production and ease of manipulation at the genetic level (Huston et al. Methods Enzymol. 203:46, 1991; Johnson, S. and Bird, R. E. Methods Enzymol. 203:88, 1991). In addition, sFv-toxin fusion proteins have been shown to exhibit enhanced anti-tumor activity in comparison with conventional chemically cross-linked conjugates (Chaudhary et al. Nature 339:394, 1989; Batra et al. Cell. Biol. 11:2200-2295, 1991). Among the first sFv to be generated were molecules capable of binding haptens (Bird et al. Science 242:423, 1988; Huston et l. Proc. Natl. Acad. Sci. USA 85:5879, 1988), cell-surface receptors (Chaudhary et al., 1989), and tumor antigens (Chaudhary et al. Proc. Natl. Acad. Sci. USA 87:1066, 1990; Colcher et al., 1990).
The gene encoding an sFv may be assembled in one of two ways: (i) by de novo construction from chemically synthesized overlapping oligonucleotides, or (ii) by polymerase chain reaction (PCR)-based cloning of V.sub.L and V.sub.H genes from hybridoma cDNA. The main disadvantages of the first approach are the considerable expense involved in oligonucleotide synthesis, and the fact that the sequence of V.sub.L and V.sub.H must be known before gene assembly is possible. Consequently, the majority of the sFv reported to date were generated by cloning from hybridoma cDNA; nevertheless, this approach also has inherent disadvantages, because it requires availability of the parent hybridoma or myeloma cell line, and problems are often encountered when attempting to retrieve the correct V region genes from heterologous cDNA. For example, hybridomas in which the immortalizing fusion partner is derived from MOPC-21 may express a V.sub.L kappa transcript which is aberrantly rearranged at the VJ recombination site, and which therefore encodes a non-functional light chain (Cabilly & Riggs, 1985; Carroll et al., 1988). Cellular levels of this transcript may exceed that generated from the productive V.sub.L gene, so that a large proportion of the product on PCR amplification of hybridoma cDNA will not encode a functional light chain. A second disadvantage of the PCR-based method, frequently encountered by the inventors, is the variable success of recovering V.sub.H genes using the conditions so far reported in the literature, presumably because the number of mismatches between primers and the target sequence destabilizes the hybrid to an extent which inhibits PCR amplification.
One method of targeting specific cells has been to make fusion proteins of a toxin and a single chain antibody. Such methods have been difficult to practice because of the difficulties in obtaining single chain antibodies and other targeting moieties. Also, none of the proposed treatment methods has been fully successful, because of the need to fuse the toxin to the targeting moiety, thus disrupting either the toxin function or the targeting function.
Thus, there exists a need for a method of providing a target cell population with a particular activity to treat tumors and other diseases.